Double-color ELISA based on fluorescence hybrid chain reaction for simultaneous detection of PEDV and PoRV-A.
📄 英文摘要 English Abstract
Porcine epidemic diarrhea virus (PEDV) and group A porcine rotavirus (PoRV-A) are two predominant pathogens leading to viral diarrhea in piglets. These two viruses often result in clinical mixed infections and exhibit indistinguishable clinical symptoms and pathological lesions, making them difficult to differentiate in routine diagnosis. Conventional ELISA suffers from low detection sensitivity and single-pathogen detection, while multiplex nucleic acid diagnostic methods rely on cumbersome nucleic acid extraction, restricting their on-site application in grassroots pig farms. Herein, an extraction-free double-color fluorescent HCR-ELISA platform is constructed for the simultaneous and ultrasensitive detection of PEDV and PoRV-A. A novel freeze-thaw combined electrostatic adsorption strategy was developed to prepare bifunctional AuNPs-Initiator-mAb probes, which rapidly integrate viral antigen recognition and HCR cascade signal initiation on a single nanoparticle. Two sets of spectrally isolated FAM/Cy3-labeled HCR hairpin probes were designed to realize crosstalk-free dual fluorescent signal amplification. The established method displayed no cross-reactivity with six common swine pathogenic viruses, with limits of detection (LOD) of 1.3 × 101 TCID50/mL for most PEDV strains and 3.2 × 101 TCID50/mL for predominant PoRV-A genotypes, and intra- and inter-batch coefficients of variation (CV) were all less than 5%, reflecting favorable specificity, sensitivity and reproducibility. Clinical detection of 191 pig fecal samples achieved total diagnostic coincidence rates of 96.3% (PEDV) and 97.9% (PoRV-A) compared with commercial RT-qPCR. Integrating enzyme-free isothermal HCR amplification with high-throughput ELISA, the double-color HCR-ELISA overcomes the limitations of traditional detection methods and possesses great promise for high-throughput screening of swine viral diarrhea in grassroots pig farms.
📄 中文摘要 Chinese Abstract
📋 英文结构化总结 English Structured Summary
摘要整理
Background:
Porcine epidemic diarrhea virus (PEDV) and group A porcine rotavirus (PoRV-A) are two predominant pathogens leading to viral diarrhea in piglets. These two viruses often result in clinical mixed infections and exhibit indistinguishable clinical symptoms and pathological lesions, making them difficult to differentiate in routine diagnosis. Conventional ELISA suffers from low detection sensitivity and single-pathogen detection, while multiplex nucleic acid diagnostic methods rely on cumbersome nucleic acid extraction, restricting their on-site application in grassroots pig farms.
Methods:
An extraction-free double-color fluorescent HCR-ELISA platform was constructed for the simultaneous and ultrasensitive detection of PEDV and PoRV-A. A novel freeze-thaw combined electrostatic adsorption strategy was developed to prepare bifunctional AuNPs-Initiator-mAb probes, which rapidly integrate viral antigen recognition and HCR cascade signal initiation on a single nanoparticle. Two sets of spectrally isolated FAM/Cy3-labeled HCR hairpin probes were designed to realize crosstalk-free dual fluorescent signal amplification.
Results:
The established method displayed no cross-reactivity with six common swine pathogenic viruses, with limits of detection (LOD) of 1.3 × 101 TCID50/mL for most PEDV strains and 3.2 × 101 TCID50/mL for predominant PoRV-A genotypes. Intra- and inter-batch coefficients of variation (CV) were all less than 5%, reflecting favorable specificity, sensitivity and reproducibility. Clinical detection of 191 pig fecal samples achieved total diagnostic coincidence rates of 96.3% (PEDV) and 97.9% (PoRV-A) compared with commercial RT-qPCR.
Data Summary:
The method showed limits of detection (LOD) of 1.3 × 101 TCID50/mL for most PEDV strains and 3.2 × 101 TCID50/mL for predominant PoRV-A genotypes, with intra- and inter-batch coefficients of variation (CV) all less than 5%. In clinical detection of 191 pig fecal samples, total diagnostic coincidence rates were 96.3% for PEDV and 97.9% for PoRV-A compared with commercial RT-qPCR.
Conclusions:
Integrating enzyme-free isothermal HCR amplification with high-throughput ELISA, the double-color HCR-ELISA overcomes the limitations of traditional detection methods. The platform possesses great promise for high-throughput screening of swine viral diarrhea in grassroots pig farms.
Practical Significance:
The extraction-free double-color fluorescent HCR-ELISA platform enables simultaneous and ultrasensitive detection of PEDV and PoRV-A without cumbersome nucleic acid extraction, supporting on-site application and high-throughput screening of swine viral diarrhea in grassroots pig farms.
📋 中文结构化总结 Chinese Structured Summary
背景:
猪流行性腹泻病毒（PEDV）和A群猪轮状病毒（PoRV-A）是导致仔猪病毒性腹泻的两种主要病原体。这两种病毒常引起临床混合感染，且临床症状和病理病变难以区分，导致常规诊断中难以鉴别。常规ELISA检测灵敏度低且只能检测单一病原体，而多重核酸检测方法依赖繁琐的核酸提取，限制了其在基层猪场的现场应用。
方法:
构建了一种免提取双色荧光HCR-ELISA平台，用于同步超灵敏检测PEDV和PoRV-A。开发了一种新型冻融结合静电吸附策略，制备双功能AuNPs-Initiator-mAb探针，该探针可在单个纳米颗粒上快速整合病毒抗原识别与HCR级联信号启动功能。设计了两套光谱分离的FAM/Cy3标记HCR发夹探针，以实现无串扰的双荧光信号放大。
结果:
所建立的方法与6种常见猪病原病毒无交叉反应，对大多数PEDV毒株的检出限（LOD）为1.3 × 10¹ TCID50/mL，对主要流行PoRV-A基因型的检出限为3.2 × 10¹ TCID50/mL。批内和批间变异系数（CV）均小于5%，表明该方法具有良好的特异性、灵敏度和重复性。对191份猪粪便样本进行临床检测，与商品化RT-qPCR相比，PEDV和PoRV-A的总诊断符合率分别为96.3%和97.9%。
数据摘要:
该方法的检出限（LOD）对大多数PEDV毒株为1.3 × 10¹ TCID50/mL，对主要流行PoRV-A基因型为3.2 × 10¹ TCID50/mL，批内和批间变异系数（CV）均小于5%。在191份猪粪便样本的临床检测中，与商品化RT-qPCR相比，PEDV和PoRV-A的总诊断符合率分别为96.3%和97.9%。
结论:
将无酶等温HCR扩增与高通量ELISA相结合，双色HCR-ELISA克服了传统检测方法的局限性。该平台在基层猪场猪病毒性腹泻的高通量筛查方面具有广阔应用前景。
实践意义:
免提取双色荧光HCR-ELISA平台无需繁琐的核酸提取即可实现PEDV和PoRV-A的同步超灵敏检测，支持基层猪场猪病毒性腹泻的现场应用和高通量筛查。